The nicotinic acetylcholine (ACh) receptors convert the binding of ACh into the opening of a cation-specific channel. The long-term goal of this project is to understand the function of the receptors in terms of their molecular structure. The overall function can be separated into three sub-functions: ACh binding, channel opening and closing, and cation-conduction. Each sub-function is associated with a type of site: the ACh binding sites, the gate and the channel. The proposed research aims at identifying, and locating in the receptor structure, the amino acid residues that form each of these sites. The channel is formed by mostly hydrophobic, membrane-spanning segments of the five receptor subunits (alpha2betagammadelta). The only residues in these segments that are accessible to water and to ions are those that line the channel lumen. To identify the channel-lining residues, most of the residues in the membrane-spanning segments of the alpha subunit will be mutated, one at a time, to cysteine. The mutant alpha, together with wild-type beta, gamma, and delta, will be expressed in heterologous cells. Mutants with near-normal function will be probed with charged, highly-water soluble, lipid-insoluble reagents which are small enough to enter the channel and which react rapidly and specifically with sulfhydryls. The reaction of these reagents with engineered cysteines in the channel will be detected electrophysiologically as an irreversible block of the ACh-induced conduction. In the cation-specific channel, positively charged reagents will react much faster than negatively charged reagents. The pattern of exposure of consecutive residues will indicate their secondary structure. The distance between pairs of engineered cysteines and their mutual exposure in the channel lumen will be probed with a positively charged bifunctional reagent. The gate will be located by probing the closed channel from both ends. The residues lining the ACh binding sites will be identified by a similar approach. Starting with the few residues already associated with these sites, individual consecutive residues will be mutated to cysteine. Those that line the water-accessible surfaces of the ACh binding sites will react with the reagents, more rapidly with the positively charged ones. Reactions at the binding site will block ACh binding and be retarded in the presence of ACh. Pairs of engineered cysteines will be crosslinkable across the binding site. Thus, a map of these sites can be achieved.